Heteropolymeric protein production methods

Abstract

Cultured mammalian cells transfected with new vectors comprising full-length or partial α and β subunit genomic DNA sequences produce significantly higher levels of dimeric glycoprotein hormone than do cells transfected with α and β subunit cDNA sequences. In cases where only the cDNA clones are available, the cDNA sequences can be used in new expression vectors comprising introns or other important genomic regions from a homologous or heterologous source.

What is claimed is: 
     
       1. A method for accumulating, in a host cell, mRNA encoding the α-subunit of a dimeric hormone selected from the group consisting of luteinizing hormone, follicle stimulating hormone, chorionic gonadotropin and thyroid stimulating hormone, comprising: transforming the host cell with a vector comprising a promoter, a structural gene encoding the α-subunit, and a terminating sequence being operatively linked to permit expression of said gene by the host cell, wherein said structural gene comprises a coding region and at least one intron, wherein one intron is immediately 5' to the coding region of the α-subunit, with the proviso that the structural gene encoding the α-subunit, is not the entire genomic sequence of the gene encoding the α-subunit of bovine luteinizing hormone; and   culturing said transformed cells under conditions by which mRNA encoding said α-subunit is produced,   wherein said intron immediately 5' to the coding region of the α-subunit is spatially disposed with respect to the ATG of the coding region such that an amount of mRNA encoding said α-subunit is produced which is greater than that which can be produced under comparable conditions using a structural gene encoding the α-subunit which is the cDNA without introns.   
     
     
       2. A method in accordance with claim 1 wherein said structural gene includes two introns. 
     
     
       3. A method in accordance with claim 1 wherein said structural gene is a cDNA encoding the α-subunit. 
     
     
       4. A method in accordance with claim 1 wherein said intron immediately 5' to the coding region is a homologous intron. 
     
     
       5. A method in accordance with claim 1 wherein said intron immediately 5' to the coding region is a heterologous intron. 
     
     
       6. A method in accordance with claim 1, wherein said structural gene encoding the α-subunit is not the entire genomic sequence of the gene encoding said α-subunit. 
     
     
       7. A method in accordance with claim 1, wherein, in the structural gene encoding the α-subunit, said one intron which is immediately 5' to the coding region of the α-subunit naturally occurs at that position in the genomic sequence of the gene encoding the α-subunit of the natural dimeric protein corresponding to the dimeric protein being produced. 
     
     
       8. A method in accordance with claim 1, wherein said structural gene contains only a single intron.